The main goal of this research proposal is to determine the mechanism of action of recombinant human vascular endothelial growth inhibitor (VEGI) as a potential anticancer therapeutic agent. VEGI is a member of the TNF superfamily (TNFSF15) we discovered. VEGI induces apoptosis in proliferating endothelial cells and growth arrest in quiescent endothelial cells. Overexpression of VEGI in tumors inhibits tumor growth in breast, prostate, and colon cancer models. We recently identified a new VEGI isoform, VEGI-192, and produced the isoform as a recombinant protein in large quantities for preclinical studies. We found that systemic administration of VEGI-192 markedly inhibited tumorigenesis and the growth of established tumors in animal models. The treatment resulted in a specific elimination of endothelial cells in the tumors, a substantial increase in the blood levels of many cytokines that mobilize the immune system, a markedly enhanced number of lymphocytes in the spleen, and a significant mobilization of hematopoietic progenitor cells in the bone marrow. In vitro VEGI was able to co-stimulate natural killer (NK) cells with interleukin-2, and induce activation and maturation of dendritic cells (DC). The identity of the VEGI receptor remains unclear. Death receptor-3 (DR3, TNFRSF25), a TNF receptor superfamily member, appears to be a candidate. DR3 has 11 differential splicing variants, three of which are transmembrane receptors, and the rest are putative soluble receptors. Expression profiles of these variants in different cell types suggest regulation of responsiveness to VEGI by these receptor variants. We hypothesize that the tumor suppression function of VEGI is mediated in part by DC and NK cells. Additionally, we hypothesize that VEGI is able to inhibit endothelial progenitor cell-supported post-natal vasculogenesis in tumors, a critical issue in tumor neovascularization. Furthermore, we hypothesize that VEGI activity is regulated by the DR3 isoforms. We propose three specific aims: 1) Determine whether VEGI mediates tumor suppression via activation of DC and NK cells;2) Determine what impact VEGI has on endothelial progenitor cell-supported vasculogenesis in tumors;and 3) whether VEGI activity is regulated by the DR3 variants.